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Physical activity guidelines targeting different populations with and without chronic diseases or disabilities are required to meet the diverse functional and physiological needs experienced by different subgroups of people to achieve optimal health benefits. As the importance of physical activity guidelines in promoting optimal health and well-being becomes increasingly recognised, there is a critical need for their systematic evaluation to ensure they remain effective, applicable and aligned with evolving health needs and scientific insights. This study aims to systematically review, critically evaluate, and compare global physical activity and sedentary behaviour guidelines on frequency, intensity, time, and type of exercise for adults, pregnant and postpartum women, and people living with chronic conditions and/or disabilities. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols checklist. We will search the Allied and Complementary Medicine Database, APA PsycInfo, Cumulative Index of Nursing and Allied Health Literature, Cochrane Library, Education Resources Information Center, Google Scholar, MEDLINE, PubMed, Scopus, SPORTDiscus, Web of Science and grey literature databases from 2010 to October 2024. Two reviewers will independently select guidelines, extract data and assess methodological quality using the Appraisal of Guidelines for Research and Evaluation II Instrument. Key recommendations will be summarised and classified as 'strong' and 'conditional' based on established criteria. A comprehensive evaluation of current guidelines will identify their differences and similarities and reveal their relevance in practical settings. The findings will guide healthcare professionals, researchers and policymakers in implementing evidence-based recommendations for managing physical activity and sedentary behaviour in targeted populations. Additionally, we will highlight current knowledge gaps and potential shortcomings in existing guidelines. PROSPERO registration number: CRD42023491339.